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. 2000 Jul;38(7):2756-9.
doi: 10.1128/JCM.38.7.2756-2759.2000.

Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene

J Pietilä  1 , Q He, J Oksi, M K Viljanen
Affiliations

Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene

J Pietilä et al. J Clin Microbiol. 2000 Jul.

Abstract

To differentiate the Borrelia burgdorferi sensu lato genospecies, LightCycler real-time PCR was used for the fluorescence (SYBR Green I) melting curve analysis of borrelial recA gene PCR products. The specific melting temperature analyzed is a function of the GC/AT ratio, length, and nucleotide sequence of the amplified product. A total of 32 DNA samples were tested. Of them three were isolated from B. burgdorferi reference strains and 16 were isolated from B. burgdorferi strains cultured from Ixodes ricinus ticks; 13 were directly isolated from nine human biopsy specimens and four I. ricinus tick midguts. The melting temperature of B. garinii was 2 degrees C lower than that of B. burgdorferi sensu stricto and B. afzelii. Melting curve analysis offers a rapid alternative for identification and detection of B. burgdorferi sensu lato genospecies.

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Figures

FIG. 1
FIG. 1
Using the real-time PCR, a fragment of the recA gene was amplified from the genospecies of B. burgdorferi sensu lato, and fluorescence melting curve analyses as well as gel electrophoresis of the products were done. (A) The Tm was 81.62°C for B. garinii, 84.02°C for B. burgdorferi sensu stricto, and 83.71°C for B. afzelii. Primer-dimers in the negative control melted at temperatures below 80°C. (B) Lane L contains molecular weight markers (100 bp), and lanes 1 to 4 show PCR products obtained from B. garinii, B. burgdorferi sensu stricto, B. afzelii, and the negative control, respectively.
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